ISOLATION AND CHARACTERISTIC OF NITROGEN-FIXING BACTERIA AND PHOSPHATE-SOLUBILIZING BACTERIA FROM SOIL HIGH IN MERCURY IN TAILINGS AND COMPOST AREAS OF ARTISANAL GOLD MINE

  AGRIVITA VOLUME 37 No. 1 FEBRUARY - 2015

  ISSN : 0126-0537

  

ISOLATION AND CHARACTERISTIC OF NITROGEN-FIXING BACTERIA

AND PHOSPHATE-SOLUBILIZING BACTERIA FROM SOIL HIGH IN MERCURY

IN TAILINGS AND COMPOST AREAS OF ARTISANAL GOLD MINE

  Brawijaya University, Malang 65145, Indonesia ₂₎ Faculty of Agriculture, Malang Islamic University Jl. M.T. Haryono, Malang 65144, Indonesia ₃₎ Faculty of Agriculture, Brawijaya University, Jl. Veteran, Malang 65145, Indonesia. _*)

  10 ¹² on seven days of incubation.

  Especially in terms of action

  Permanent vegetation cover is known as effective and environmentally friendly way to turn mine tailings and to trigger the process of soil formation (Moreno et al., 2004).

  The results of the study reported by several investigators show that mine waste containing mercury has negative effect on seedling, growth, biochemical properties and the results of some crops (Munzuroglu and Geekil, 2002; Sekhar et al., 2005).

  Acidiphilium, while the previously dominant Legionella becomes completely undetectable.

  Mine tailings are usually characterized by a high content of heavy metals due to the use of heavy metals in the process of separating the gold from the rich solution. In addition, it is also characterized by the content of organic matter, nitrogen and phosphorus which are very low, neutral to acidic, and highly potential for generating acid. Tailing heaps generally do not have the hard soil structure and overgrown plants (Krzaklewski and Pietrzykowski, 2002). Tailings cause stress on the microbial community. Ryan et al. (2013) implied that the main species found are of the genera Acidithiobacillus and

  Mining on various types of minerals in various countries are now common, including in Indonesia. In Indonesia, there are currently five mine. Freeport gold and copper mine has doubled, and the nickel mine Indo (Sulawesi) has embarked on the expansion of its operations. Although re-vegetation efforts on the tailings area have been done, none of the mines disposed of tailings correctly. So that the potential long-term impact of these activities cannot be certainly known (McMahon et al., 2000).

  INTRODUCTION

  Keywords: N-fixing bacteria, P-solubilizing bacteria, mercury, phytoremediation

  % molasses medium with the number of 0.15 x

  Corresponding author Phone: +62 341-553623 E-mail: yulianuraini@yahoo.com

  pumilus and Bacillus megaterium effective at 5

  Yulia Nuraini ^{1), 3*)} , Novi Arfarita ^{1), 2)} and Bambang Siswanto ^{1), 3)} ₁₎ International Research Centre for the Management of Degraded and Mining Lands,

  Pseudomonas, and N-fixing bacteria found was Pseudomonas and Azotobacter. While testing

  the waste corn and peanuts that has been composted for 4 weeks acquired P-solubilizing bacteria, such as Enterobacter, Bacillus and

  Bacillus megaterium and Bacillus pumilus. While

  10 ³ cfu g ^-1 . Identification of species using Becton Dickinson Phoenix test, both species belong to

  The research was conducted at Brawijaya University and West Nusa Tenggara, from March 2013 to October 2013. The tailings areas of the gold mine contains high mercury (Hg) as much as 1,090 ppm, and living microbes (resistance) exist in a small number in such a condition. Microbial P solvents encountered came from the genus Bacillus with a population of 23 x 10 ³ cfu g ^-1 and N-fixing bacteria encountered are of the genus Bacillus, with a population of 4 x

  

Received: June 19, 2014/ Accepted: January 28, 2015

ABSTRACT

  the activity and antagonism of N-fixing and P- solubilizing bacteria, the result on agar media did not show antagonism in its growth. Bacillus

  Yulia Nuraini et al.: Isolation and Characteristic of Nitrogen-Fixing Bacteria and Phosphate- Solubilizing Bacteria….…. the root zone (Rascio and Navari-Izzo, 2011). The use of native plants (indigenous) is the focus of this phyto-stabilization technology because native plants are generally tolerant to local environmental conditions and provide a foundation for the natural ecological viability.

  Compost material used in this research was the rest of the legumes crop, corn and cattle manure (Table 1). Each crop residue was obtained from 500 kg of agricultural land in the vicinity in Senggigi, Lombok, West Nusa Tenggara, Indonesia. The rest of the plants was collected and chopped into the size of ± 2 cm. Each type of crop residue was then mixed with cow dung with a ratio of 20:1 (5% cow dung). Then 250 kg of each mixture was dissolved in

  Microbial population was determined using Total Plate Count on N fix semisolid medium, followed by further observations on colony morphology. Observation of cell morphology was performed with gram staining. N-fixing was then measured on the N fix semisolid medium.

  10 ^-2 , 10 ^-3 , 10 ^-4 , 10 ^-5 , 10 ^-6 , 10 ^-7 ) ml ml ^-1 (v/v). Isolation of non-symbiotic N fixing bacteria was performed using N fix semisolid media.

  Isolation, selection and identification to obtain bacterial isolates of N-fixing and P- solubilizing used legume compost, corn compost and tailings, from which 100 gr was taken and shaken with 90 ml of 0.85% NaCl for 30 minutes. Furthermore, the clear solution was then dissolved into seven concentrations (10 ^-1 ,

  Bacterial Isolation, Selection and Identification

  C. If the temperature reached more than 50°C and the mixture was stirred again. Composting process was stopped when the temperature had stabilized at 28 ^o C and the color turned into black.

  50 L of water. Once dissolved, the mixture was dumped and covered with thick plastic for 30 days. During the period of composting, the temperature was observed to maintain the temperature below 50 ^o

  Composting

  In addition to re-vegetation technique to support reclamation or mining heap in the gold mine, the role of N-fixing bacteria and P solubilizing bacteria after phytoremediation of mercury in the tailing areas of gold mine was proposed in this study. The use of indigenous species and strains that have been adapted with local soil and climatic conditions is highly recommended. Utilization of local plants and indigenous bacteria is suggested so that the growth of vegetation and microbes can help restore and stabilize the environment. In the long term, it is expected that the microclimate can be improved, the biodiversity can be restored, and improve soil conditions towards more productive. N-non symbiotic fixing bacteria (Azospirillim sp, Azotobacter sp, Aerosomonas

  This study was conducted from March 2013 to October 2013 in the Laboratory of areas of Artisanal gold mine, Lombok, West Nusa Tenggara.

  MATERIALS AND METHODS Research Sites

  The purpose of this study was to isolate the non-symbiotic N-fixing bacteria and P solubilizing bacteria from local compost and to determine the effect of various propagation culture media of bacteria. Besides, this research also studied the effect of the application of non- symbiotic N-fixing bacteria and P-solubilizing bacteria after phytoremediation mercury in gold mine tailings using indicator plant growth and microbial biodiversity.

  Aspergillus sp, Penicillium, and Pseudomonas sp (Mikanova and Novaka, 2002).

  aggregate stability. Strain bacteria isolation and selection on acid soil Azotobacter is also able to produce growth hormone regulation besides its N-Fixing potential (Widayati, 1998). P solubilizing bacteria can produce enzyme phosphatase, as well as organic acid which produces citrate, oxalate, and succinate (Paul and Clark, 1989). Spesies microorganism P solubilizing as like

  sp and Aspergillus sp) can also fungtion as soil

  Bacterial identifications were performed using microbiological tests (morphology and biochemistry). Samples of bacteria were isolated and cultured in a nutrient medium and medium specific order. Identification of biochemical tests was performed with system identification automatically name the Becton Dickinson Phoenix Instrument Test, commonly known as BD Phoenix Test. Yulia Nuraini et al.: Isolation and Characteristic of Nitrogen-Fixing Bacteria and Phosphate- Solubilizing Bacteria….…. parameter soil and bacteria N fixing and P- solubilizing. Observations on the compost included pH, C/N ratio, and total N Fixing bacteria, P-solubilizing bacteria and total microorganisms. Further test activity to find out bacteria that had been selected was done by using Picovskaya (pour plate) and N-semisolid medium (MPN).

  RESULTS AND DISCUSSION Analysis of Soil Hg in Senggigi Region

  2.10 C/N Kjeldahl dan Walkey Black

  pH (H ₂ O) H ₂ O

  7.20

  8.00 pH (KCl) KCl

  7.00

  7.90 C-Organik (%) Walkey Black

  34.28

  22.83 N-total (%) Kjeldahl

  1.83

  19.00

  Remarks: td = not ditected (0) Table 2. Chemical characteristic of analyzed corn and legume waste

  11.00 P-total (%) HNO3+HCO4

  0.21

  0.81 K-total (%) HNO3+HCO4

  1.06

  1.73 Table 3. Compost characteristic of analyzed corn and legume

  Compost pH C/N N-available (ppm) P-available (ppm) Total Microorganism (cfu/ml 10 ⁷ ) H ^{2 O KCl NH 4 NH 3} Corn

  8.20

  8.10 8 562 176 830

  Analysis Method Corn Waste Legume Waste

  11.68 Pb (ppm) Td 530 Fe (ppm) Td 3,810 Mn (ppm) Td 4,840 Zn (ppm) Td 3,760

  The results of the soil analysis showed that the content of Hg contaminated soil was quite high, 1,090 (ppm) (Table 1(b)). Soil pH was neutral but tended to be alkali (base), C- organic low and low CEC. It is very dangerous for the health of animals or humans when they enter the food chain obtained from water or any food contaminated with the Hg. Thus, the high Hg levels need to be lowered to the normal range (Table 1(a)). This can be done by using a heavy metal accumulator plants (phyto- remediation), which has been proven to reduce or absorb Hg soil. However, it is known there are microorganisms and organic material that can reduce the levels of heavy metals or decompose into harmless compounds. The use of beneficial microorganisms (potential) and compost can degrade heavy metals as well as adding nutrients, especially N and P required.

  2.89 S (ppm)

  Table 1. Results of physical and chemical analy- sis of soil and tailings in people gold mine locations district of Senggigi, West Lombok

  Soil Properties and Unit Un- contaminated Soil by Tailing (a) Contaminated Soil by Tailing (b)

  Texture Silty loam Sandy loam pH

  6.40

  7.70 C organic (%)

  0.95

  1.19 N total (%) 0.10 0.001 P-available (ppm)

  0.98

  8.92 CEC (cmol/kg)

  4.0 Hg (ppm) Td 1,090 Au (ppm) Td

  14.25

  11.57 K-dd (cmol/kg) 3.25 0.001 Ca- exchangable (cmol/kg)

  3.04

  1.99 Mg- exchangable (cmol/kg)

  1.26

  0.84 Na- exchangable (cmol/kg)

  0.89

  0.64 Cu (ppm) Td 792 Cd (ppm) Td

  40

  Yulia Nuraini et al.: Isolation and Characteristic of Nitrogen-Fixing Bacteria and Phosphate- Solubilizing Bacteria….….

  Microbial Analysis of Soil Tailing

  Analysis of soil taken from a gold mine Clear zone was detected because the disposal process that contained mercury (Hg) solubilizing Ca-P with organic acid was produced was found high for its Hg level (1,090 ppm) and measured by the clear zone index from (Table 1). In such a condition, N-Fixing and P- diameter colony and size of colony. Expression solubilizing bacteria were found but only in a of the dissolution of P could be measured by small number (Table 6). N-fixing bacteria after solubilizing P. Nitrogen fixation expressed purification was obtained only 1 species and amount of N fixing (N2), which showed ₃ quantity of 4 x 10 cfu/g soil while P-solubilizing bacteria’s ability to fix Nitrogen in the air (Husen bacteria although the number was the same but et al., 2007). ₃ the total population was larger, 23 x 10 cfu/g soil (Table 4). The lack of bacteria because of Table 5. Bacterial capability in fixed N and higher mercury could stress the growth of dissolved P microbacteria especially the growth of N fixing

  Dissolved P

  and P solubilizing (Thangavel and Subhuram,

  Fixed N Clear 2004). No Isolate Dissolved (ppm) Zone P (ppm) Index

  Table 4. Bacterial characteristic of Hg contami-

  1 L2P1 1 1.4 -

  3.7 nated soil (tailing)

  2 L2P2 1

  1.8 5.3 -

  Microbial

  2.8

  0.4 Bacterial Cell Gram

• 3 L2P2 2

  No Total

• _-1 Type Form Staining

  1.5

  5.0

• 4 M2P1 2

  (cfu g ) ³

  5 M2P3 1

  1.0

  0.1 -

  1 N-fixing 4 x 10 Bacillus positive ³

  6 M2P3 2

  1.3

  3.5 -

  2 P-solubilizing 23 x 10 Bacillus positive

  7 L2N1 - -

  8 Bacterial Capability in N Fixation and

• 8 L2N2 -

  94

  dissolved P

  9 - M2N1 90 - Bacterial activity of dissolved P and N 10 - - M2N2

  32 fixation activity was listed in Table 5. Each bacterium had different activity in the dissolved P

  Remarks: - = not detected and N fixation. Dissolved P were characterized by the formation of a clear zone around the colonies of bacteria and it could quantify the amount of dissolved P and N fixed bacteria.

  Table 6. The result of bacterial screening process from Hg-contaminated Soil (Tailling)

  Soil Parameter Unit Microbial Total Method Sample

• _-1
₃ MPN (Most

  Tailing Non-symbiotic N Fixing bacteria (N1 and N2) cfu gr 4 x 10

  Probable Number) from

• _-1
₃ Lombok P Solubilizing bacteria (P1, P2 and P3) cfu gr 23 x 10 Pour plate
Yulia Nuraini et al.: Isolation and Characteristic of Nitrogen-Fixing Bacteria and Phosphate- Solubilizing Bacteria….…. Table 7. Becton Dickinson Phoenix instrument test of bacterial strain N1

  Instru- Instru- Instru- Bio- Expected Bio- Expected Bio- Expected No ment No ment No ment chemical Result chemical Result chemical Result Result Result Result

  1 A_ARARR

  17 A_GLPRB - 32 A_LALT +

  2 A_LARGH

  18 A_LHIST - 33 A_LISO - - - - -

  19 A_LPHET

  34 A_LPROB - + +

• 3 A_LLEUH -

  4 A_LPYR - 20 A_LTRY - 35 A_META - - -

  5 C_3MGA + +

  21 C_CLST v

  36 C_DFRU + + +

  6 C_DGUA

  22 C_DMNT

  37 C_IMN v + + + + +

  7 C_KGA

  23 C_MAA v + 38 C_PXB

  8 C_THY

  24 M_ADGLU + 39 M_BDCEL +

• v

  9 M_BDGAL

  25 M_BDGLC - 40 M_BDGLU + - + - -

  10 M_NAG

  26 M_PHOS

  41 M\PHOT + - + + v

• 11 N_ALALH

  27 N_LPROT

  42 N_VAALA - - - - - -

  12 P_ADGLU -

  43 R_BGEN - - - -

• 28 P_PHOL

  13 R_DEX

  29 R_DSUC - 44 R_DTAG -

  30 R_MAL - 45 R_MTT

• 14 R_DTRE

  15 R_NGU

  31 S_URE

  46 T_ESC v + - - - -

  16 R_MGP v - Table 8. Becton Dickinson Phoenix instrument test of bacterial strain P2

  Instru- Instru- Instru- Bio- Expected Bio- Expected Bio- Expected No ment No ment No ment chemical Result chemical Result chemical Result Result Result Result

  1 A_ARARR v -

  17 A_GLPRB

  32 A_LALT v - - -

• 2 A_LARGH

  18 A_LHIST

  33 A_LISO - - - - v

  3 A_LLEUH

• v

  19 A_LPHET

  34 A_LPROB -

  4 A_LPYR

  20 A_LTRY - 35 A_META -

• v

  5 C_3MGA

  21 C_CLST - 36 C_DFRU + + - + +

  6 C_DGUA +

  22 C_DMNT

  37 C_IMN + + +

  7 C_KGA

  23 C_MAA + 38 C_PXB

• 39 M_BDCEL

  24 M_ADGLU

• 8 C_THY

  9 M_BDGAL

  25 M_BDGLC + 40 M_BDGLU +

  10 M_NAG v

  26 M_PHOS v + 41 M\PHOT + + -

  11 N_ALALH v

  27 N_LPROT

  42 N_VAALA v - + + -

  28 P_PHOL v + 43 R_BGEN

• 12 P_ADGLU
• 13 R_DEX

  29 R_DSUC v

  44 R_DTAG - - + +

• 30 R_MAL v -
• 15 R_NGU

  45 R_MTT v

• 14 R_DTRE

  31 S_URE

  46 T_ESC v + - - - - Yulia Nuraini et al.: Isolation and Characteristic of Nitrogen-Fixing Bacteria and Phosphate- Solubilizing Bacteria……..

  Bacterial Selection and Identification

  No Incubation (Day) Total Ammount of Bacteria Colony (cfu/g) Media

  pumilus and Bacillus megaterium was 0.5 x

  Mercury polluted areas in the gold mine showed fairly high levels (1,090 ppm) and these need to be reduced to a non-dangerous level. Still there were microbes that were tolerant in spite of their small numbers. N fixing bacterium obtained was Bacillus pumillus with total amount of 4 x 10 ³ cfu g ^-1 soil, and P solubilizing bacteria obtained was Bacillus megaterium with a population greater than that of N fixing bacteria (23 x 10 ³ cfu g ^-1 ). Nitrogen fixing bacteria populations of corn and legume compost have a larger population. It was expected that mixing bacteria and compost reduced Hg concentration bound by carboxyl groups and phenolic of compost. P would be available in the orthophosphate, able to bind Hg and increase P nutrient supply. N fixing bacteria could increase the fertility of soil contaminated with Hg. Antagonist interaction did not occur between the bacteria. The optimum population of mixture of Bacillus

  CONCLUSION

  3 9 x 10 ⁵ Molase 5% 3 8 x 10 ⁹ Molase 5% 7 0.15 x 10 ¹² Molase 5%

  2 4 x 10 ⁵ Molase 10% 3 1.67 x 10 ¹⁰ Molase 10% 7 2.88 x 10 ¹² Molase 10%

  1 7 x 10 ⁵ NB 3 11.15 x 10 ⁹ NB 7 2.14 x 10 ¹² NB

  Table 9. Bacterial growth in liquid culture media (Molasses and Nutrient Broth)

  Selected and indentified P-solubilizing and N-fixing bacteria were taken from soil contaminated with Hg. Two isolates of N1 and N2 were obtained from Non-symbiotic N fixing on MPN medium (Table 6). Of P-solubilizing bacterial were obtained 3 isolates and named P1, P2 and P3 (Table 6). N1 and P2 were then selected to be used in further study. Bacterial identification using Becton Dickinson Phoenix test showed that N1 belong to Bacilus pumilus (Table 7) and P2 belong to Bacillus magetarium (Table 8).

  After 7 days of incubation, all liquid carrier media showed good results. Both media mically more efficient because it produced bacterial population of 0.15 x 10 ¹² cfu/g. This amount was almost equal to the 10% molasses and NB. Molasses was used as a source of energy C for microbes to grow.

  The application of N fixing bacteria and P solubilizing bacteria selected (N1 and P2) were then tested for their ability to grow in a liquid carrier medium (Nutrient Broth) and molasses in concentration of 5% and 10% as shown in Table 9.

  Effect of Liquid Carrier on The Growth of Bacteria

  Bacteria isolated from soil contaminated with Hg (Tailling) and have the ability in N fixation was N1 and N2 and the P dissolution. Three isolates namely P1, P2, P3 were taken from P dissolution. They were then put together on Nutrient Agar to determine the interaction between the bacterial antagonisms. No inhibition zone was found among the five bacteria. Instead, the bacteria were proven to grow synergistically. Furthermore, they could be applied simultaneously as mixed culture.

  Antagonist Test of Isolated Bacteria

  P dissolution activity test of bacteria isolated performed clear zone around bacterial colonies. The wider the clear zone provided the more P (H ^{2 PO 4 ) that can be used by plants. P 2} was the highest in dissolved P and has been identified as Bacillus megaterium. N fixing bacteria was marked by a color change from green to blue to N-fix semisolid medium, in which N1 was the highest in N-fixation and had been identified as Bacillus pumilus.

  Microbial Activity Test in Phosphate Dissolution and N Fixation

  10 ¹² , found on 5% moles. Yulia Nuraini et al.: Isolation and Characteristic of Nitrogen-Fixing Bacteria and Phosphate- Solubilizing Bacteria….…. General of Higher Education for the funding the implementation of this study. Thanks are also expressed to those who have assisted in the completion of this study.

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